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Neuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.

DOI： 10.1016/j.isci.2018.05.019

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DOI： 10.1016/j.neures.2017.11.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. Toexaminehowsignaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

DOI： 10.1523/JNEUROSCI.3319-16.2017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Euglenoid flagellates have striped surface structures comprising pellicles, which allow the cell shape to vary from rigid to flexible during the characteristic movement of the flagellates. In Euglena gracilis, the pellicular strip membranes are covered with paracrystalline arrays of a major integral membrane protein, IP39, a putative four-membrane-spanning protein with the conserved sequence motif of the PMP-22/EMP/MP20/Claudin superfamily. Here we report the three-dimensional structure of Euglena IP39 determined by electron crystallography. Two-dimensional crystals of IP39 appear to form a striated pattern of antiparallel double-rows in which trimeric IP39 units are longitudinally polymerised, resulting in continuously extending zigzag-shaped lines. Structural analysis revealed an asymmetric molecular arrangement in the trimer, and suggested that at least four different interactions between neighbouring protomers are involved. A combination of such multiple interactions would be important for linear strand formation of membrane proteins in a lipid bilayer.

DOI： 10.1038/ncomms2731

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The effect of subtype-selective phosphodiesterase (PDE) inhibitors on acid secretion was examined in mouse stomachs to investigate which PDE isozymes are involved in the local regulation of this secretion. Male DDY mice were used after 18 h fasting. An isolated stomach was incubated in an organ bath containing buffered solution gassed with 95% O(2)/5% CO(2), while the lumen was perfused with unbuffered solution gassed with 100% O(2). Acid secretion was measured at pH 5.4 using a pH-stat method. Histamine or pituitary adenylate cyclase activating polypeptide (PACAP) was added to the serosal solution. PDE inhibitors were added to the serosal solution 30 min before histamine or PACAP. The secretion of acid in the isolated stomach was increased by histamine or PACAP, and these responses were totally inhibited by famotidine. IBMX alone increased basal acid secretion and significantly enhanced the acid responses to histamine and PACAP. Among the PDE inhibitors tested, only rolipram (PDE4 inhibitor) significantly increased basal acid secretion and potentiated the acid responses to histamine and PACAP. The latter peptide increased histamine release into the medium, and this response was also enhanced by rolipram. Furthermore, rolipram significantly increased cAMP production induced in the isolated stomach by histamine and PACAP. These results suggest that PDE4 is involved in the local regulation of gastric acid secretion via the degradation of cAMP and that the PDE4 inhibitor rolipram increases the secretion of acid by potentiating acid production in parietal cells and enhancing histamine release from enterochromaffin-like cells.

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The role of nitric oxide synthase (NOS) isozymes in the aggravation of indomethacin-induced gastric damage in adjuvant arthritic rats was investigated. Two weeks after injection of Freund's complete adjuvant, the animals were given indomethacin, and the stomach was examined for damage 4 h later. Indomethacin caused hemorrhagic lesions in the normal rat stomach, and these lesions were markedly aggravated in arthritic rats. Pretreatment with L-NAME (a nonselective inhibitor of NOS) and aminoguanidine (a relative selective inhibitor of iNOS) did not affect the ulcerogenic response in normal rats but dose-dependently prevented the aggravation of lesions in arthritic rats, but the effect of aminoguanidine was apparently less than that of L-NAME. The increased ulcerogenic response in arthritic rats was significantly suppressed by 1400 W (a selective inhibitor of iNOS) and L-NIO (a selective inhibitor of eNOS) but not by L-NPA (a selective inhibitor of nNOS). The concurrent administration of 1400 W and L-NIO almost totally abolished the aggravation of damage in arthritic rats. The expressions of eNOS and iNOS but not nNOS in the gastric mucosa were clearly enhanced in arthritic rats. Mucosal levels of non-protein sulfhydryls were significantly lower in arthritic rats than those in normal rats. The aggravation of damage in arthritic rats was significantly prevented by glutathione. These results suggest that the increased ulcerogenic response to indomethacin in arthritic rat stomachs is mediated by NO derived from eNOS in addition to iNOS. It is assumed that eNOS/NO may act harmfully on the gastric mucosa of arthritic rats with mucosal SH deficiency.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Aims: We investigate the role of nitric oxide (NO) in the hypersecretion of acid and pepsinogen induced by stomach distension.
Main method: The rat stomach was distended by instillation of saline through an acute fistula under urethane anesthesia.
Key findings: Both secretions of acid and pepsinogen were increased by the distension depending on the volume of saline introduced, and responses were attenuated by bilateral cervical vagotomy or prior administration of atropine. N(G)-nitro-L-arginine methyl ester (L-NAME) had a dual effect on these responses, causing an increase in the acid response and a decrease in the pepsin response, both in an L-arginine-sensitive manner. Distension of the stomach increased the luminal NO release; this response was suppressed by vagotomy and L-NAME. Intragastric application of FK409, a NO donor, dose-dependently increased pepsinogen secretion while decreasing acid secretion in the stomach without distension. However, serosal application of both FK409 and 8-bromo-guanosine cyclic 3', 5'-monophosphate (8-Br-cGMP) stimulated the secretion of pepsinogen in isolated mouse stomachs in vitro. The stimulatory effect of FK409 on pepsinogen secretion was totally abolished by LY83583, a guanylate cyclase inhibitor.
Significance: Distension of the stomach increases both acid and pepsinogen secretion through a vagalcholinergic pathway in addition to the luminal release of NO, and NO affects these responses in opposite ways, suppressing the acid response while enhancing the pepsin response, both mediated by a guanylate cyclase/cGMP pathway. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2008.10.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

NSAIDs damage the small intestine as well as the stomach as adverse effects. We previously reported that the gastric ulcerogenic response to NSAIDs was markedly increased in arthritic rats. The present study was designed to examine the intestinal ulcerogenic property of indomethacin in adjuvant-induced arthritic rats in comparison with normal animals. Arthritis was induced in male Dark Agouti rats by injection of Freund's complete adjuvant into the right hindfoot. Two weeks later, indomethacin was given orally and the intestine was examined for lesions at several time points after indomethacin. Indomethacin produced intestinal lesions in both normal and arthritic rats, but in the latter, the ulcerogenic response occurred much earlier and the severity was markedly enhanced. Amino, guanidine, an inhibitor of iNOS, significantly suppressed the damage, yet the efficacy differed in normal and arthritic rats, depending on the dose schedule; the effect of post-administration (6 h after) was greater than that of pre-administration (0.5 h before) in normal rats, whereas that of post-administration was less than that of pre-administration in arthritic rats. The expression of iNOS and TLR4 in the intestine was enhanced in arthritic rats as compared with normal rats. These results suggest that the intestinal ulcerogenic response to indomethacin is markedly aggravated in arthritic rats. Notably, the onset of the ulceration was much earlier in arthritic rats than normal rats. These phenomena may be accounted for by the upregulation of iNOS/NO through the increased expression of TLR4 in the small intestine of arthritic rats. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2007.08.036

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